1. Volume 21, Issue 9, Pages 2515-2527 (November 2017)
Receptor Activator of NF-κB Orchestrates Activation of Antiviral Memory CD8 T Cells in the Spleen Marginal Zone 
Mohamed Habbeddine, Christophe Verthuy, Olivia Rastoin, Lionel Chasson, Magali Bebien, Marc Bajenoff, Sahil Adriouch, Joke M.M. den Haan, Josef M. Penninger, Toby Lawrence 
Cell Reports 
Volume 21, Issue 9, Pages (November 2017)
DOI: /j.celrep
Copyright © 2017 The Author(s) Terms and Conditions

2. Cell Reports 2017 21, 2515-2527DOI: (10.1016/j.celrep.2017.10.111)
Copyright © 2017 The Author(s) Terms and Conditions

3. Figure 1 RANK Expression Does Not Affect DC Survival or Longevity
(A) Gating strategy for RANK+ DCs; representative fluorescence-activated cell sorting (FACS) plots are shown.
(B) Proportion of RANK+ DCs (MHC-IIhi CD11c+ cells) among immune cells in different organs (CLN, cutaneous LN; MLN, mesenteric LN; PP, Peyer’s patches).
(C) Number of RANK+ DCs in CLN from Tnfrsf11af/f and Tnfrsf11aΔItgax mice.
(D) Total numbers of DCs in LN and spleen.
(E) Proportions of DC1, DC2, and pDC subsets in CLN and spleen.
(F) Relative numbers of resident DCs (resDC) and migratory DCs (migDC) in CLN.
(G) Lethally irradiated CD45.1+ mice were reconstituted with a 1:1 mix of bone marrow cells from either Tnfrsf11af/f or Tnfrsf11aΔItgax mice (donor; CD45.2+) and wild-type cells from CD45.2+/CD45.1+ mice (competitor). BrdU incorporation by MHC-IIhi CD11c+ cells was measured in CLN from chimeric mice by flow cytometry; the ratio of BrdU+ resDCs and migDCs from donor (Tnfrsf11af/f and Tnfrsf11aΔItgax) and competitor cells is shown.
(H) Proportion of RANK+ DCs in CLN from wild-type mice with or without IMQ treatment.
(I) Proportion of RANK+ DCs in CLN from Tnfrsf11af/f and Tnfrsf11aΔItgax mice after IMQ treatment; representative FACS plots are shown.
(J) Quantification of total DC numbers in CLN from Tnfrsf11af/f and Tnfrsf11aΔItgax mice 2 days after IMQ treatment.
Data are represented as mean ± SEM of n ≥ 3 and are representative of at least 2 independent experiments.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2017 The Author(s) Terms and Conditions

4. Figure 2
RANK Expression by CD11c+ Cells Regulates mCTL Activation in Response to Viral Infection
(A and B) Expansion of endogenous OVA-specific CD8 T cells (Tet-H2KbSIINFEKL; Tet+) was measured in peripheral blood from Tnfrsf11af/f and Tnfrsf11aΔItgax mice 1 week after infection i.v. with 1 × 104 CFUs of Lm-OVA (A) or 1 × 105 PFUs of VSV-OVA (B). IFNγ production was measured by intracellular cytokine staining (ICS) after ex vivo stimulation with cognate peptide (SIINFEKL).
(C–F) Cohorts of Tnfrsf11af/f and Tnfrsf11aΔItgax mice were immunized with Lm-OVA, IMQ/OVA, or VSV-OVA; (C) Lm → VSV, (D) IMQ → VSV, (E) Lm → Lm, and (F) VSV → Lm. 2 months later, mice were challenged with either VSV-OVA (C and D) or Lm-OVA (E and F). 5 days later, expansion of OVA-specific mCTLs (CD44+ Tet+) was measured in spleen by flow cytometry.
(G–I) Shown in (G): experimental strategy for the adoptive transfer of mCTLs. CD45.1+ mice were immunized with Lm-OVA; 3 weeks later, CD4 and CD8 T cells were isolated from spleen and adoptively transferred to cohorts of naive Tnfrsf11af/f and Tnfrsf11aΔItgax mice. A further 7 weeks after T cell transfer, mice were infected with VSV-OVA (H) or Lm-OVA (I); activation of adoptively transferred mCTLs (CD44+, CD45.1+) and priming of endogenous naive CD8 T cells (CD45.2+) were measured simultaneously in spleen 5 days after challenge as expansion of Tet+ cells.
Data are represented as mean ± SEM, and statistical analysis was performed with the Mann-Whitney test; ∗∗p < 0.01.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2017 The Author(s) Terms and Conditions

5. Figure 3 RANK Is Required for CD11c+ CD169+ MZMs
(A–D) Immunohistochemical analysis of spleen sections from Tnfrsf11af/f and Tnfrsf11aΔItgax mice analyzed by confocal microscopy. (A) RANKL staining (red) of marginal reticular stromal cells (MRCs) in the spleen marginal zone (MZ); CD4 staining for T cells is in blue. (B) Non-specific esterase staining of spleen sections. (C) CD169 (green; filled arrowheads) and CD209b staining (red; open arrowheads); B cells are stained with B220 (blue). (D) Quantification of CD169, CD209b, and B220 staining in the spleen MZ of Tnfrsf11af/f and Tnfrsf11aΔItgax mice. Data are represented as the mean intensity (±SEM) within the MZ from n ≥ 3 mice.
(E) Spleen sections from Rosa26-LSL-tdRFP × Tg(Itgax-Cre) mice showing Cre-recombinase activity in CD169+ cells; red indicates tdRFP, and green indicates CD169.
(F) Spleen sections from Tg(Itgax-EYFP) mice showing CD11c expression in CD169+ cells; blue indicates YFP, green indicates CD169, and red indicates CD209b. Scale bars: 100 μm in the upper panels and 25 μm in the lower panels. Representative micrographs are shown from at least 2 independent experiments.
Statistical analysis was performed with the Mann-Whitney test; ∗∗∗∗p <
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2017 The Author(s) Terms and Conditions

6. Figure 4 RANK-Dependent MZMs Provide a Niche for VSV Replication
(A and B) Lm-OVA-immunized mice were infected i.v. with VSV-OVA, and spleens were harvested after 7 hr for analysis of viral replication. (A) Splenic titers of VSV-OVA from immunized Tnfrsf11af/f and Tnfrsf11aΔItgax mice. (B) qRT-PCR analysis of OVA expression in total splenocytes.
(C and D) Lm-OVA-immunized Tnfrsf11af/f and Tnfrsf11aΔItgax mice were challenged with VSV-GFP. (C) Confocal microscopy of spleen sections 7 hr after infection with VSV-GFP; viral replication (green) is shown in CD169+ cells in the MZ (red); B cells are stained with B220 (blue). Scale bars: 100 μm in the upper panels and 25 μm in the lower panels. Representative micrographs are shown from at least 2 independent experiments. (D) Quantification of GFP+ CD169+ cells in the MZ.
(E) Splenic titers of VSV-GFP from Tnfrsf11af/f and Tnfrsf11aΔItgax mice 7 hr after infection.
(F and G) Flow cytometry analysis of GFP expression in CD8α+ splenic DCs from Lm-OVA-immunized Tnfrsf11af/f and Tnfrsf11aΔItgax mice 7 hr after VSV-GFP infection. Gating strategy (F) and quantification (G) are shown.
Representative FACS plots are shown, and graphs represent mean ± SEM of n ≥ 6. Statistical analysis was performed with the Mann-Whitney test. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p <
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2017 The Author(s) Terms and Conditions

7. Figure 5 RANK-Dependent MZMs Are Not Sufficient for mCTL Activation
(A–C) Immunohistochemical analysis of spleen sections from Tnfrsf11af/f and Tnfrsf11aΔLyz2 mice analyzed by confocal microscopy; (A) Non-specific esterase staining of spleen sections. (B) CD169 (green; filled arrowheads) and CD209b staining (red; open arrowheads); B cells are stained with B220 (blue). Scale bars: 100 μm. Representative micrographs are shown from at least 2 independent experiments. (C) Quantification of CD169, CD209b, and B220 staining in the spleen MZ of Tnfrsf11af/f and Tnfrsf11aΔLyz2 mice; data are represented as the mean intensity (±SEM) within the MZ from n ≥ 3 mice.
(D) Tnfrsf11af/f and Tnfrsf11aΔLyz2 mice were infected i.v. with 106 PFUs of VSV-GFP, and spleens were harvested after 7 hr for analysis of viral replication.
(E) Expansion of OVA-specific CD8 T cells (Tet+) was measured in blood from Tnfrsf11af/f and Tnfrsf11aΔLyz2 mice 8 days after infection i.v. with Lm-OVA or VSV-OVA.
(F) Cohorts of Tnfrsf11af/f and Tnfrsf11aΔLyz2 mice were immunized with Lm-OVA and, 2 months later, challenged i.v. with VSV-OVA; expansion of OVA-specific mCTLs (CD44+ Tet+) was measured 5 days later in spleen by flow cytometry.
Graphs represent mean ± SEM of n = 5. Statistical analysis was performed with Mann-Whitney test. ∗p < 0.05; ∗∗∗∗p <
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2017 The Author(s) Terms and Conditions

8. Figure 6 Collaboration of MZMs and DC1 for Activation of mCTLs
(A) Frequency of RANK+ DC in spleen 7 hr after primary or secondary VSV challenge in Tnfrsf11af/f and Tnfrsf11aΔItgax mice.
(B) Naive or Lm-OVA-immunized Tnfrsf11af/f and Tnfrsf11aΔItgax mice were infected with VSV-OVA; 7 hr after infection, induction of CXCL9 and IFNγ mRNA expression were measured in total splenocytes by qRT-PCR. Data are expressed as fold induction compared to uninfected mice (Ctl) and are indicated as the mean ± SEM of n = 7–9.
(C) Localization of CXCL9+ DCs (green/white; filled arrowheads) in the spleen MZ, marked by RANKL staining (red/pink; open arrowheads), in Lm-OVA-immunized Tnfrsf11af/f and Tnfrsf11aΔItgax mice 7 hr after infection with VSV-OVA.
(D) CXCL9 induction in splenic DCs was measured by intracellular flow cytometry; representative FACS plots are shown, and graph indicates mean ± SEM.
(E) OT-I-GFP cells were adoptively transferred to cohorts of Tnfrsf11af/f and Tnfrsf11aΔItgax mice prior to immunization with Lm-OVA. 2 months later, mice were infected with VSV-OVA, and spleens were collected after 7 hr for visualization of mCTL mobilization by confocal microscopy; recruitment of memory OT-I-GFP cells (green; asterisk) in the spleen MZ is indicated, juxtaposed to RANKL+ MRCs (red; open arrowheads) and CD11c+ DCs (blue; closed arrowheads). Scale bars: 100 μm in the upper panels and 25 μm in the lower panels. Representative micrographs are shown from at least 2 independent experiments. Bar graph indicates quantification of OT-I-GFP clusters in Tnfrsf11af/f and Tnfrsf11aΔItgax mice 7 hr after infection with VSV-OVA.
(F) Wild-type mice were immunized with Lm-OVA and, 4 months later, challenged i.v. with wild-type VSV and administration i.p. of either OVA-conjugated anti-DEC205, anti-DCIR2, or isotype-control antibody. Five days later, endogenous OVA-specific mCTL (CD44+ Tet+) expansion was measured in spleen by flow cytometry.
(G) Lm-OVA-immunized Tnfrsf11af/f and Tnfrsf11aΔItgax mice were challenged with wild-type VSV with OVA-conjugated anti-DEC205 or isotype-control antibody; mCTL expansion was measured in spleen after 5 days.
Graphs represent mean ± SEM of n = 2–6 for isotype control and n = 5–7 for the other groups. Statistical analysis was performed with the Mann-Whitney test. ∗∗p < 0.01; ∗∗∗p <
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2017 The Author(s) Terms and Conditions